The c-Myb transcription factor is a key regulator of hematopoietic cell differentiation, proliferation and apoptosis and expression of the c-myb gene is essential for normal hematopoiesis. However, a few minor mutations unleash the latent oncogenic activity of c-Myb, giving it the ability to transform hematopoietic cells in tissue culture and induce leukemias and lymphomas in chickens and mice. Furthermore, alternative RNA splicing produces at least a dozen different variants of human c-Myb protein with dramatically different activities. The alternative splicing is much more frequent in leukemias and appears to be an important mechanism for generating oncogenic variants of c-Myb in human tumors. Specific Aim 1 will compare the transcriptional and biological activities of the wild type and variant forms of c-Myb produced by normal and leukemic cells. Wild type and variant Myb proteins will be expressed in normal CD34+ hematopoietic progenitor cells, then microarrays will detect changes in gene expression and colony formation and other cell-based assays will measure changes in proliferation, differentiation or apoptosis. By correlating these results, the relationship between specific changes in c-Myb protein structure and its transcriptional and biological functions will be determined. Specific Aim 2 will use proven and novel models of leukemic cell transformation to directly test whether wild type and variant forms of c-Myb are able to transform immature hematopoietic cells to a leukemia-like phenotype in tissue culture. The results will be used to identify the changes in c-Myb that unleash its oncogenic activity. Specific Aim 3 will determine how minor changes in c-Myb structure cause such dramatic changes in activity. Structure-function experiments will determine whether wild type c-Myb or variants produced by alternative RNA splicing have altered interactions with co-factors or other proteins or altered stability or sub- cellular localization. The goal will be to understand how minor changes in structure affect c-Myb activity at the biochemical level. The goal of this renewal application is to understand how relatively minor changes in the structure of the c-Myb protein can lead to such dramatic changes in its activity, from normal regulator to oncoprotein, and to characterize mechanisms that could activate the oncogenic activity of c-Myb in human tumors.